Modulation of sterol homeostasis by the Cdc42p effectors
Cla4p and Ste20p in the yeast Saccharomyces cerevisiae
Meng Lin
1,
*, Karlheinz Grillitsch
2,
*, Gu
¨
nther Daum
2
, Ursula Just
1
and Thomas Ho
¨
fken
1
1 Institute of Biochemistry, Christian Albrecht University, Kiel, Germany
2 Institute of Biochemistry, Graz University of Technology, Austria
Keywords
cell polarity; p21-activated kinase; sterol;
steryl ester; yeast
Correspondence
T. Ho
¨
fken, Institute of Biochemistry,
Christian Albrecht University Kiel,
Olshausenstrasse 40, 24098 Kiel, Germany
Fax: +49 431 8802609
Tel.: +49 431 8801660
E-mail: [email protected]
*These authors contributed equally to this

l
MINT-7291480: STE20 (uniprotkb:Q03497) physically interacts (MI:0915) with BEM1
(uniprotkb:
P29366)byubiquitin reconstruction (MI:0112)
l
MINT-7291468: STE20 (uniprotkb:Q03497) physically interacts (MI:0915) with NCP1
(uniprotkb:
P16603)byubiquitin reconstruction (MI:0112)
l
MINT-7291441: STE20 (uniprotkb:Q03497) physically interacts (MI:0915) with ERG4
(uniprotkb:
P25340)byubiquitin reconstruction (MI:0112)
l
MINT-7291492: CLA4 (uniprotkb:P48562) physically interacts (MI:0915) with BEM1
(uniprotkb:
P29366)byubiquitin reconstruction (MI:0112)
l
MINT-7291412: STE20 (uniprotkb:Q03497) physically interacts (MI:0915) with ARE1
(uniprotkb:
P25628)bypull down (MI:0096)
l
MINT-7291424: STE20 (uniprotkb:Q03497) physically interacts (MI:0915) with ARE2
(uniprotkb:
P53629)bypull down (MI:0096)
Abbreviations
GST, glutathione S-transferase; PAK, p21-activated kinase; SC, synthetic complete; SE, steryl esters; YPD, 1% yeast extract, 2% peptone,
2% dextrose.
FEBS Journal 276 (2009) 7253–7264 ª 2009 The Authors Journal compilation ª 2009 FEBS 7253
Introduction
The Rho-type GTPase Cdc42p plays a crucial role in

and exit [16,17]. Ste20p activates mitogen-activated
protein kinase cascades controlling mating, filamentous
growth and the hyperosmotic stress response [18–22].
Ste20p also contributes to mitotic exit and cell death
[16,23]. Furthermore, Cla4p and Ste20p are both
involved in vacuolar inheritance [24].
Previously, we have demonstrated that Ste20p binds
to Erg4p, Cbr1p and Ncp1p, which are all involved in
sterol biosynthesis [25]. We also observed genetic inter-
actions between PAKs and ERG4 as well as between
PAKs and NCP1. Both ERG4 and NCP1 are essential
in the cla4D background. Furthermore, STE20 deletion
exacerbates the growth defect of the ncp1 D strain [25].
Cells lacking either ERG4 or NCP1 exhibit defects in
bud site selection, apical bud growth, cell wall assem-
bly, mating, filamentous growth and mitotic exit [25–
27]. Notably, Ste20p and Cla4p also play important
roles in these processes. No phenotypic changes were
observed for the cbr1D strain. By contrast, inactivation
of CBR1 and NCP1 results in lethality. The large
majority of these cells have abnormal bud morphology
[25]. Other groups also reported a role for sterols in
mating [28,29] and it has been suggested that sterol
biosynthesis may increase during formation of a mating
projection [28]. Furthermore, homologues of oxysterol-
binding proteins, a family of proteins that regulate
the synthesis and transport of sterols, were found to
participate in Cdc42p-dependent polarity [30]. Taken
together, these observations suggest that sterol synthe-
sis may play a crucial role in cell polarization and in

tion by Ste20p [37]. Therefore, it is conceivable that
Ste20p may regulate the activity of this SE-synthesiz-
ing enzyme. Considering the importance of sterols for
cell polarization, and the interactions between PAKs
and proteins catalyzing sterol synthesis and storage, it
is tempting to speculate that Ste20p and Cla4p may
influence sterol homeostasis. In this work, we show
that sterol levels are increased in cells lacking either
STE20 or CLA4. The absence of CLA4 also leads to
higher amounts of SE. Furthermore, CLA4 expression
from a multicopy plasmid results in reduced activity of
Are2p, the major enzyme of SE formation under aero-
bic conditions. These data suggest that Ste20p and
Cla4p may negatively influence sterol homeostasis.
Sterol homeostasis modulation by Ste20p and Cla4p M. Lin et al.
7254 FEBS Journal 276 (2009) 7253–7264 ª 2009 The Authors Journal compilation ª 2009 FEBS
Results
Cells lacking either STE20 or CLA4 exhibit
increased sterol levels
Sterol biosynthesis plays an important role in cell
polarization [25,26,28,29]. Here, we examined whether
the Cdc42p effectors Ste20p and Cla4p contribute to
the regulation of sterol biosynthesis. To achieve this,
lipids were extracted from the wild-type yeast and cells
lacking either CLA4 or STE20 and sterols were ana-
lyzed using GLC ⁄ MS. All major sterols were increased
in both the ste20D and the cla4D mutants (Table 1). In
these deletion strains, the amounts of ergosterol and
total sterols were approximately 1.3-fold higher com-
pared with those in the wild-type strain (P < 0.05).

were grown in selective medium, in contrast to the
strains analyzed in Table 1, which were incubated in
YPD medium. The different composition of these
types of media probably accounts for the difference in
sterol levels.
GLC ⁄ MS, employed here, not only determines the
amount of free unesterified sterols in membranes but
also the amount of sterols derived from SE that are
Table 1. Sterol analysis of cells lacking STE20 and CLA4. Data are mean values with standard deviation from at least two independent
experiments.
lg of sterol per mg of protein
wild-type ste20D cla4D swe1D cla4D swe1D
Ergosterol 15.21 ± 0.66 20.1 ± 1.67 19.58 ± 0.57 15.15 ± 0.98 18.18 ± 1.11
Zymosterol 0.62 ± 0.14 0.84 ± 0.06 0.76 ± 0.11 0.73 ± 0.06 1.14 ± 0.31
Fecosterol 0.48 ± 0.16 0.64 ± 0.04 0.65 ± 0.07 0.45 ± 0.04 0.89 ± 0.30
Lanosterol 0.28 ± 0.09 0.45 ± 0.15 0.38 ± 0.06 0.26 ± 0.08 0.35 ± 0.07
Total sterol 16.59 ± 0.59 22.03 ± 1.65 21.37 ± 0.78 16.49 ± 1.09 20.56 ± 1.52
A
B
Fig. 1. Cell morphology of the strains used
in this study. (A) Morphology of deletion
strains. The indicated strains were grown in
YPD to stationary phase. The cells were
then fixed with formaldehyde and examined
by microscopy. Bars: 5 lm. (B) Expression
of either STE20 or CLA4 from a multicopy
plasmid does not affect cell morphology.
Cells were grown in minimal medium to
stationary phase. Bars: 5 lm.
M. Lin et al. Sterol homeostasis modulation by Ste20p and Cla4p

(Table 3). Thus, pheromone signaling does not seem to
have an effect on biosynthesis of the major sterols.
Cla4p negatively influences SE formation
We also examined the potential link between the
Cdc42 effectors Ste20p and Cla4p and the SE syn-
thases Are1p and Are2p. To start with, it was tested
(using a pull-down assay) whether Ste20p forms a
complex with Are1p. Indeed, epitope-tagged Are1p
expressed in yeast bound specifically to recombinant
Ste20p from Escherichia coli (Fig. 4). Are2p, the major
SE synthase under aerobic conditions, also interacted
Table 2. Sterol analysis of cells overexpressing STE20 and CLA4.
Data are mean values with standard deviation from at least two
independent experiments.
lg of sterol per mg of protein
pRS425 pRS425-STE20 pRS425-CLA4
Ergosterol 26.67 ± 2.12 21.32 ± 0.86 27.44 ± 0.90
Zymosterol 2.36 ± 0.11 1.99 ± 0.16 2.53 ± 0.22
Fecosterol 0.92 ± 0.05 0.91 ± 0.09 1.20 ± 0.08
Lanosterol 0.60 ± 0.27 0.31 ± 0.10 0.68 ± 0.10
Total sterol 30.55 ± 2.10 24.53 ± 1.01 31.85 ± 1.11
Fig. 2. Cells lacking either STE20 or CLA4 have increased levels of
free sterol. The indicated strains were grown to stationary phase,
and then lipids were extracted and separated by TLC. The data
shown are from two independent experiments. *, P < 0.05 com-
pared with the wild–type strain.
A
B
Fig. 3. Cla4p does not bind to Erg4p, Cbr1p or Ncp1p. (A) The
split-ubiquitin system. The N-terminal and C-terminal halves of

ases, we specifically tested whether Are1p and Are2p
have a role in cell polarity. Bud site selection, mating
and filamentous growth was normal in cells lacking
ARE1 and ARE2 (data not shown), but apical bud
growth following G
1
cyclin overexpression was affected
(Fig. 5). During budding, the cyclin-dependent kinase
Cdc28p promotes polarized apical growth when
coupled to the G
1
cyclins and isotropic growth when
associated with mitotic cyclins [47]. The apical growth
phase can be prolonged by G
1
cyclin overexpression,
resulting in hyperelongated buds [47] (Fig. 5A,B). Cells
deleted for genes encoding cell-polarity proteins, such
as Ste20p, form fewer hyperpolarized buds in response
to overexpression of the G
1
cyclin CLN1 [25,48]
(Fig. 5B). To test whether Are1p and Are2p are
involved in apical bud growth, we overexpressed
CLN1 in the corresponding deletion strains and scored
for the presence of hyperelongated buds. The deletion
of either ARE1 or ARE2 resulted in a smaller number
of cells with an elongated bud (Fig. 5B). A further
decrease was observed for the are1D are2D double
mutant. Immunoblot analysis revealed that the mutant

CLA4 expression from a multicopy plasmid led to a
marked decrease of Are2p enzyme activity (Fig. 7B).
Taken together, these data suggest that Cla4p nega-
tively influences sterol biosynthesis and storage.
Discussion
Sterols play an important, but ill-defined, role in cell
polarity [25,28–30]. It has been suggested that sterol
Table 3. Sterol composition of cells in response to a-factor. Data
are expressed as mean values with standard deviation from five
independent experiments.
lg of sterol per mg of protein
Dimethylsulfoxide a-factor
Ergosterol 21.89 ± 2.00 20.84 ± 1.61
Zymosterol 1.20 ± 0.14 1.24 ± 0.20
Fecosterol 0.48 ± 0.15 0.49 ± 0.15
Lanosterol 0.61 ± 0.32 0.55 ± 0.29
Total sterol 24.18 ± 1.94 23.12 ± 1.71
Fig. 4. Ste20p interacts with both SE synthases.Purified GST and
GST-Ste20p were immobilized on glutathione-sepharose beads
and incubated with a yeast lysate of ARE1-9myc, ARE2-9myc or
CYC8-9myc cells. Eluted proteins were analyzed by immunoblotting
using anti-myc IgG. One per cent of the input is shown. Predicted
molecular mass values: Are1p-9myc, 81 kDa; Are2p-9myc, 83 kDa;
Cyc8p-9myc, 116 kDa.
M. Lin et al. Sterol homeostasis modulation by Ste20p and Cla4p
FEBS Journal 276 (2009) 7253–7264 ª 2009 The Authors Journal compilation ª 2009 FEBS 7257
synthesis might increase during polarization [25,28]
and that Cdc42p effectors, such as Cla4p and Ste20p,
may control sterol biosynthesis [25]. In this work, we
showed that cells lacking either STE20 or CLA4 have

could influence sterol synthesis. In contrast to Ste20p,
Cla4p does not bind to Erg4p, Cbr1p and Ncp1.
We also showed here that sterol levels during polari-
zation in response to a-factor treatment remain con-
stant. Ste20p is essential for the arrest at G
1
and the
formation of a mating projection following pheromone
stimulation [18,20], and Cla4p also seems to play a
minor role in this pheromone signalling [56,57], but
neither protein seems to affect sterol biosynthesis dur-
ing the formation of a mating projection. Nevertheless,
the phenotypes of mutants defective in ergosterol
synthesis clearly demonstrate the importance of sterols
for polarization during mating [25,28,29]. On the
other hand, the observation that sterols enrich at the
tip of mating projections, where they could anchor
polarity proteins, has been a controversial point of dis-
cussion [28,29,58,59]. Our data suggest that the forma-
tion of these sterol-rich domains does not involve a
A
B
C
Fig. 5. Are1p and Are2p have a role in api-
cal bud growth. (A) Morphology of normal
and hyperelongated cells. Exponentially
growing cells carrying pGAL1-CLN1-3HA on
a plasmid were induced by the addition of
galactose for 4 h. The cells were then fixed
with formaldehyde. (B) Are1p and Are2p are

tive effect, not only on sterol biosynthesis but also on
SE formation. Reduced Are2p activity in cells contain-
ing multicopy CLA4 does not affect the levels of SE.
Possibly, the amount of Are2p in the cell is relatively
high in relation to its substrate. A reduction of enzyme
activity would then not necessarily have an effect on
SE levels. Alternatively, it may simply take more time
to form SE. In contrast to CLA4, STE20 deletion and
expression from a multicopy plasmid, respectively, had
no effect on either SE levels or SE synthase activity.
Nevertheless, Ste20p phosphorylates Are2p [37] and we
show here that Ste20p forms a complex with Are1p
and Are2p. The functional link behind this finding is
not clear.
Interestingly, Ste20p and Cla4p also down-regulate
sterol uptake by inhibiting the expression of genes
involved in this process (Lin and Ho
¨
fken, manuscript
submitted). Therefore, it seems that Ste20p and Cla4p
negatively influence several important sterol homeo-
static events. Sterol homeostasis is critical for the cell
and is linked to cell polarization. The importance of
sterol biosynthesis for polarization during vegetative
growth, mating and filamentation has previously been
demonstrated [25,28]. In this study, we showed that the
SE synthases Are1p and Are2p are also involved in api-
cal bud growth during G
1
cyclin overexpression. Taken

carrying either STE20 or CLA4 on a multicopy plasmid, or the vec-
tor alone, were treated as described in panel A. Data are from two
independent experiments. *, P < 0.05 compared with the wild-type
strain.
M. Lin et al. Sterol homeostasis modulation by Ste20p and Cla4p
FEBS Journal 276 (2009) 7253–7264 ª 2009 The Authors Journal compilation ª 2009 FEBS 7259
way in secretory vesicles and in the plasma membrane
during bud formation and growth.
Our data also raise the question of how sterols con-
tribute to cell polarization at the molecular level. Two
mechanisms are conceivable. Sterols have a crucial
function in endocytosis [61], which in turn is required
for the establishment and maintenance of cell polarity
(e.g. by counteracting lateral diffusion of polarized
proteins within the membrane) [58,62]. Alternatively,
sterols may be important in the association of proteins
involved in establishing cell polarity with the plasma
membrane, which occurs independently of endocytosis.
It has been suggested that sterol-rich domains are com-
partmentalized in the plasma membrane and serve as
an anchor for proteins involved in establishing cell
polarity [28,59]. However, the existence and biochemi-
cal nature of such domains is unclear [28,29,58,59] and
further investigations will be required to elucidate the
role of sterols in cell polarization in more detail.
Materials and methods
Yeast strains, plasmids and growth conditions
All yeast strains used in this study are in the YPH499 back-
ground and are listed in Table 4. Yeast strains were grown in
1% yeast extract, 2% peptone, 2% dextrose (YPD) medium

Myc (9E10) mAb and the rabbit polyclonal anti-Cdc11p
IgG were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Monoclonal mouse anti-HA (12CA5) was
obtained from Roche Diagnostics (Mannheim, Germany)
and peroxidase-conjugated secondary IgG was obtained
from Pierce (Rockford, IL, USA).
Pheromone response and apical growth assays
For the pheromone response assay, cells grown to the
logarithmic phase were incubated with 1 lgÆmL
)1
of a-factor
Table 4. Yeast strains used in this study.
Name Genotype
Source or
reference
MLY2 YPH499 are1D::klTRP1 This study
MLY3 YPH499 are2D::klTRP1 This study
MLY6 YPH499 are1D::klTRP1 are2D::HIS3MX6 This study
MLY20 YPH499 HIS3MX6-pGAL1-ARE2-9myc-klTRP1 This study
MLY21 YPH499 are1D::HIS3MX6 ste20D::klTRP1 This study
MLY28 YPH499 HIS3MX6-pGAL1-ARE1-9myc-klTRP1 This study
MLY84 YPH499 are1D::HIS3MX6 cla4D::kanMX6 This study
MLY115 YPH499 ARE1-9myc-HIS3MX6 This study
THY310 YPH499 ste20D::klTRP1 [25]
THY609 YPH499 cla4D::kanMX6 This study
THY665 YPH499 swe1D::HIS3MX6 This study
THY685 YPH499 swe1D::HIS3MX6 cla4D::kanMX6 This study
YPH499 MATa ura3-52 lys2-801 ade2-101
trp1D63 his3D200 leu2D1
[70]

with galactose for 4 h and fixed with 4% formaldehyde
(final concentration) for microscopic examination.
Microscopy
For microscopic examination, cells were fixed with 4%
formaldehyde (final concentration) and analyzed using a
Zeiss Axiovert 200M fluorescence microscope equipped
with a 100· Plan oil-immersion objective. Images were
captured using a Zeiss AxioCam MRm CCD camera.
Lipid extraction and analysis
Total cellular lipids were extracted as described previously
[67]. Individual sterols were identified and quantified using
GLC ⁄ MS after alkaline hydrolysis of lipid extracts [68].
The protein concentration of 10 mL of culture with an
attenuance at 600 nm of 1 was determined and cells were
incubated for 2 h at 90 °C together with 0.6 mL of metha-
nol, 0.4 mL of 0.5% pyrogallol dissolved in methanol,
0.4 mL of 60% aqueous KOH and 10 lg of cholesterol dis-
solved in ethanol as an internal standard. Lipids were
extracted three times with n-heptane and the combined
extracts were taken to dryness under a stream of nitrogen.
Then, lipids were dissolved in 10 lL of pyridine, and after
adding 10 lL of N,O-bis(trimethylsilyl)–trifluoroacetamide
(Sigma), samples were diluted with ethyl acetate to an
appropriate concentration. GLC ⁄ MS analysis of silylated
sterol adducts was performed on a Hewlett-Packard HP
5890 Series II gas chromatograph (Palo Alto, CA, USA),
equipped with an HP 5972 mass selective detector and an
HP 5-MS column (cross-linked 5% phenyl methyl siloxane;
dimensions 30 m · 0.25 mm · 0.25 lm film thickness).
Aliquots of 1 lL were injected in the splitless mode at an

trated sulfuric acid, briefly dried and heated at 100 °C for
20 min. SE were then quantified by densitometric scanning at
400 nm using a Shimadzu dual-wavelength chromatoscanner
CS930, with cholesteryl ester as the standard.
Acyl-CoA:ergosterol acyltransferase assay
The acyl-CoA:ergosterol acyltransferase assay was per-
formed in a final volume of 100 lL containing 6 nmol
of [
14
C]oleoyl-CoA (88 000 disintegrations per minute),
0.025 mm ergosterol, 0.5 mm CHAPS, 100 mm KH
2
PO
4
(pH 7.4), 1 mm dithiothreitol and 200 lg of protein from
the homogenate of cells grown to logarithmic phase [69].
This method relies on the measurement of the amount of
radiolabeled steryl esters formed during the assay relative to
the substrate employed under standardized conditions. Incu-
bations were carried out for 30 min at 30 °C and terminated
by the addition of 300 lL of chloroform ⁄ methanol (2 : 1,
v ⁄ v). Lipids were extracted twice for 10 min with shaking
using 300 lL of chloroform ⁄ methanol (2 : 1; v ⁄ v), each.
The organic phases were combined and washed twice using
methanol ⁄ water ⁄ chloroform (47 : 48 : 3, v ⁄ v ⁄ v). The extrac-
tion efficiency of the substrate formed was > 95%. The
organic phase was taken to dryness under a stream of nitro-
gen. Lipids were dissolved in 30 lL of chloroform ⁄ methanol
(2 : 1, v ⁄ v), separated by TLC (as described above) and
visualized on TLC plates by staining with iodine vapor.

lated signal transduction in Saccharomyces cerevisiae.
Mol Cell Biol 20, 7559–7571.
7 Barale S, McCusker D & Arkowitz RA (2006) Cdc42p
GDP ⁄ GTP cycling is necessary for efficient cell fusion
during yeast mating. Mol Biol Cell 17, 2824–2838.
8Mo
¨
sch HU, Roberts RL & Fink GR (1996) Ras2
signals via the Cdc42 ⁄ Ste20 ⁄ mitogen-activated protein
kinase module to induce filamentous growth in
Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93,
5352–5356.
9Mo
¨
sch HU, Ku
¨
bler E, Krappmann S, Fink GR &
Braus GH (1999) Crosstalk between the Ras2p-con-
trolled mitogen-activated protein kinase and cAMP
pathways during invasive growth of Saccharomyces
cerevisiae. Mol Biol Cell 10, 1325–1335.
10 Mo
¨
sch HU, Ko
¨
hler T & Braus GH (2001) Different
domains of the essential GTPase Cdc42p required for
growth and development of Saccharomyces cerevisiae.
Mol Cell Biol 21, 235–248.
11 Hofmann C, Shepelev M & Chernoff J (2004) The

19 Liu H, Styles CA & Fink GR (1993) Elements of the
yeast pheromone response pathway required for fila-
mentous growth of diploids. Science 262, 1741–1744.
20 Ramer SW & Davis RW (1993) A dominant truncation
allele identifies a gene, STE20, that encodes a putative
protein kinase necessary for mating in Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 90, 452–456.
21 O’Rourke SM & Herskowitz I (1998) The Hog1 MAPK
prevents cross talk between the HOG and pheromone
response MAPK pathways in Saccharomyces cerevisiae.
Genes Dev 12, 2874–2886.
22 Raitt DC, Posas F & Saito H (2000) Yeast Cdc42
GTPase and Ste20 PAK-like kinase regulate Sho1-
dependent activation of the Hog1 MAPK pathway.
EMBO J 19, 4623–4631.
23 Ahn SH, Cheung WL, Hsu JY, Diaz RL, Smith MM &
Allis CD (2005) Sterile 20 kinase phosphorylates histone
H2B at serine 10 during hydrogen peroxide-induced
apoptosis in S. cerevisiae. Cell 120, 25–36.
24 Bartholomew CR & Hardy CF (2009) p21-activated
kinases Cla4 and Ste20 regulate vacuole inheritance in
Saccharomyces cerevisiae. Eukaryot Cell 8, 560–572.
25 Tiedje C, Holland DG, Just U & Ho
¨
fken T (2007)
Proteins involved in sterol synthesis interact with Ste20
and regulate cell polarity. J Cell Sci 120, 3613–3624.
26 Ni L & Snyder M (2001) A genomic study of the
bipolar bud site selection pattern in Saccharomyces
cerevisiae. Mol Biol Cell 12, 2147–2170.

molecular species composition of yeast subcellular mem-
branes reveals acyl chain-based sorting ⁄ remodeling of
distinct molecular species en route to the plasma mem-
brane. J Cell Biol 146, 741–754.
34 Yang H, Bard M, Bruner DA, Gleeson A, Deckelbaum
RJ, Aljinovic G, Pohl TM, Rothstein R & Sturley SL
(1996) Sterol esterification in yeast: a two-gene process.
Science 272, 1353–1356.
35 Yu C, Kennedy NJ, Chang CC & Rothblatt JA (1996)
Molecular cloning and characterization of two isoforms
of Saccharomyces cerevisiae acyl-CoA:sterol acyltrans-
ferase. J Biol Chem 271, 24157–24163.
36 Zweytick D, Leitner E, Kohlwein SD, Yu C, Rothblatt
J & Daum G (2000) Contribution of Are1p and Are2p
to steryl ester synthesis in the yeast Saccharomyces
cerevisiae. Eur J Biochem 267, 1075–1082.
37 Ptacek J, Devgan G, Michaud G, Zhu H, Zhu X,
Fasolo J, Guo H, Jona G, Breitkreutz A, Sopko R
et al. (2005) Global analysis of protein phosphorylation
in yeast. Nature 438, 679–684.
38 Cvrckova F, De Virgilio C, Manser E, Pringle JR &
Nasmyth K (1995) Ste20-like protein kinases are
required for normal localization of cell growth and for
cytokinesis in budding yeast. Genes Dev 9, 1817–1830.
39 Sakchaisri K, Asano S, Yu LR, Shulewitz MJ, Park CJ,
Park JE, Cho YW, Veenstra TD, Thorner J & Lee KS
(2004) Coupling morphogenesis to mitotic entry. Proc
Natl Acad Sci USA 101, 4124–4129.
40 Longtine MS, Theesfeld CL, McMillan JN, Weaver E,
Pringle JR & Lew DJ (2000) Septin-dependent assembly

York, NY.
47 Lew DJ & Reed SI (1993) Morphogenesis in the yeast
cell cycle: regulation by Cdc28 and cyclins. J Cell Biol
120, 1305–1320.
48 Nelson B, Kurischko C, Horecka J, Mody M, Nair P,
Pratt L, Zougman A, McBroom LD, Hughes TR,
Boone C et al. (2003) RAM: a conserved signaling
network that regulates Ace2p transcriptional activity
and polarized morphogenesis. Mol Biol Cell 14, 3782–
3803.
49 Lai MH, Bard M, Pierson CA, Alexander JF, Goebl
M, Carter GT & Kirsch DR (1994) The identification
of a gene family in the Saccharomyces cerevisiae ergos-
terol biosynthesis pathway. Gene 140, 41–49.
50 Reddy VV, Kupfer D & Caspi E (1977) Mechanism of
C-5 double bond introduction in the biosynthesis of
cholesterol by rat liver microsomes. J Biol Chem 252,
2797–2801.
51 Aoyama Y, Yoshida Y, Sato R, Susani M & Ruis H
(1981) Involvement of cytochrome b5 and a cyanide-
sensitive monooxygenase in the 4-demethylation of
4,4-dimethylzymosterol by yeast microsomes. Biochim
Biophys Acta 663, 194–202.
52 Yoshida Y (1988) Cytochrome P-450 of fungi: primary
target for azole antifungal agents. In Current Topics in
Medical Mycology (McGinnis MR ed), Vol 2, pp.
388–418. Springer, New York, NY.
53 Aoyama Y, Yoshida Y, Sonoda Y & Sato Y (1989)
Deformylation of 32-oxo-24,25-dihydrolanosterol by the
purified cytochrome P-45014DM (lanosterol 14 alpha-

59 Proszynski TJ, Klemm R, Bagnat M, Gaus K & Simons
K (2006) Plasma membrane polarization during mating
in yeast cells. J Cell Biol 173, 861–866.
60 Kurat CF, Wolinski H, Petschnigg J, Kaluarachchi S,
Andrews B, Natter K & Kohlwein SD (2009)
Cdk1 ⁄ Cdc28-dependent activation of the major triacyl-
glycerol lipase Tgl4 in yeast links lipolysis to cell-cycle
progression. Mol Cell 33, 53–63.
61 Pichler H & Riezman H (2004) Where sterols are required
for endocytosis. Biochim Biophys Acta 1666, 51–61.
62 Marco E, Wedlich-Soldner R, Li R, Altschuler SJ &
Wu LF (2007) Endocytosis optimizes the dynamic local-
ization of membrane proteins that regulate cortical
polarity. Cell 129, 411–422.
63 Sherman F (1991) Getting started with yeast. In Guide to
Yeast Genetics and Molecular and Cell Biology (Guthrie
C & Fink GR eds), pp. 3–21. Elsevier, San Diego.
64 Longtine MS, McKenzie A, Demarini DJ, Shah NG,
Wach A, Brachat A, Philippsen P & Pringle JR (1998)
Additional modules for versatile and economical PCR-
based gene deletion and modification in Saccharomyces
cerevisiae. Yeast 14, 953–961.
65 Knop M, Siegers K, Pereira G, Zachariae W, Winsor
B, Nasmyth K & Schiebel E (1999) Epitope tagging of
yeast genes using a PCR-based strategy: more tags and
improved practical routines. Yeast 15, 963–972.
66 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the Folin phenol
reagent. J Biol Chem 193, 265–275.
67 Folch J, Lees M & Sloane-Stanley GH (1957) A simple